Genome-wide characterization of the Triplophysa dalaica slc4 gene family and expression profiles in response to salinity changes

Background The solute carrier 4 (SLC4) gene family is involved in a variety of physiological processes in organisms and is essential for maintaining acid-base balance in the body. The slc4 genes have been extensively studied in mammals, and they play important roles in intracellular and extracellular pH regulation and in the secretion and uptake of HCO3− and other ions (Na+ and Cl−) between transepithelial cells in different tissues. This study identified and characterized the entire slc4 gene family of Triplophysa dalaica. Results Fifteen slc4 genes were identified in the whole genome of Triplophysa dalaica in this study, including five copies of Na+-independent Cl−/HCO3− transporters, eight members of Na+-dependent HCO3− transporters, and two genes coding Na+-coupled borate transporters. The chromosomal location information, isoelectric points, and molecular weights of the 15 slc4 genes were analyzed. The results for gene structure, domain analysis, and phylogenetic relationships of this gene family showed that the slc4 genes (except for slc4a9, which is missing in teleosts) are significantly expanded in teleosts compared to higher vertebrates. This phenomenon suggests that the slc4 gene family played an important role in the transition from aquatic to terrestrial animals. RT-PCR results showed that different slc4 genes showed diversified expression patterns in the tissues of T. dalaica. For osmotic pressure regulating organs, slc4a1b, slc4a4b, slc4a7, and slc4a11a were highly expressed in gills. In the kidney, slc4a1a, slc4a3, and slc4a10b were highly expressed, suggesting that the slc4 genes play a specific role in the salinity adaptation of T. dalaica. Our study has deciphered the biological roles of the slc4 genes in maintaining ionic and acid-base homeostasis in teleost fishes and provides a foundation for future exploration of the highly differentiated gene family in Triplophysa. Conclusions The results are relevant for the breeding of alkali-tolerant varieties in saline-alkali areas for aquaculture. Our findings have important implications for the adaptation process of freshwater species to saline-alkali water. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-022-09057-8.

At present, research on the slc4 genes in fish is limited, primarily focused on a single or several members of the slc4 gene family. There is increasing evidence to suggest that the slc4 genes are closely related to the acid-base regulation and ion regulation of fishes [8,9]. For example, it has been suggested that slc4a1 and slc4a4 play key roles in the ion-regulatory pathway of zebrafish gills [10,11] suggested that slc4a1, slc4a2, and slc4a4 are involved in the alkali adaptation process in the gill and kidney of Leuciscus waleckii [12]. observed a salinity-dependent expression pattern of the slc4a4 genes in the foregut of Sparus aurata L. Studies have also shown that the slc4a4 genes are significantly induced in Takifugu obscurus [13] and Oreochromis mossambicus [14] during seawater domestication. In 2020, researchers conducted a systematic study of the slc4 gene family in Lateolabrax maculatus and found that the expression patterns of the slc4 genes in the gill tissue were different in response to alkalinity stress and salinity changes in terms of the degree of differential expression. Understanding how these closely related proteins perform different functions is therefore one of the major challenges and opportunities for studying transporter families.
T. dalaica inhabits rivers with relatively gentle water flow. It is small in size and has strong adaptability. It is widely distributed in northern China in the tributaries of the Yellow River and some artesian water bodies such as Dali Lake in Inner Mongolia. Dali Lake is located in the internal flow basin of eastern Inner Mongolia (116°39′24″E, 43°22′43″N); its alkalinity can be as high as 53.57 mmol/l, and its pH can reach 9.62 [15]. After long-term natural environment domestication, the fish composition of Dali Lake at present consists of fish with high salt tolerance such as Leuciscus waleckii, Carassius auratus Linnaeus, Pseudorasbora parva, and T. dalaica [16]. suggested that T. dalaica has the strongest resistance to salinity and alkalinity and can survive in different water quality conditions. Therefore, T. dalaica is an ideal model species for studying the mechanism of adaptation to highly saline environments. At present, there are few domestic studies on the salinity adaptability of loach [17,18]. In this study, the slc4 gene family was identified; phylogenetic and gene structure analyses were performed; and the expression of slc4 genes in different tissues of T. dalaica in freshwater and saline-alkali water habitats was examined. The expression patterns of the family members involved in salinity adaptation were explored, providing a new basis for studying the adaptation mechanisms of slc4 genes in T. dalaica to saline environments and in addition promoting the aquaculture of suitable breeding varieties in saline-alkali areas.

Gene structure analysis of slc4 genes
To compare the structural diversity of the slc4 gene family, its domains and exon-intron structures were visualized using TBtools (Fig. 1). The results showed that most members of the Cl − /HCO 3 − exchangers possessed [20][21][22] exons, except for slc4a1b that had 16 exons. Among the members of the Na + /HCO 3 − transporters, slc4a5a, slc4a5b, slc4a7, slc4a8, and slc4a10a consisted of 25 exons. There were 23, 26, and 24 exons in slc4a4a, slc4a4b, and slc4a10b, respectively. There were 15 and 19 exons, respectively, in the Na + -coupled boronate transporters slc4a11a and slc4a11b. In addition, three functional domains were predicted in the slc4 gene family, namely Band_3_cyto, HCO3_ cotransp, and PTS_EIIA_2. HCO3_cotransp, located at the C-terminus of all slc4 genes, indicating that it is highly conserved. Band_3_cyto exists in Cl − /HCO 3 − exchanger and Na + /HCO 3 − cotransporter. Band_3_ cyto is a cytoplasmic domain that associates with the cell membrane cytoskeleton proteins to stabilize the membrane skeleton. PTS_EIIA_2 was only present in slc4a11a and slc4a11b. Overall, the predicted results of the exon-intron structures and domains of the 15 slc4 genes were consistent with those of the phylogenetic tree.

Motif analysis of slc4 genes
The T. dalaica slc4 genes were searched through the MEME database, and a total of 10 conserved motifs were identified (Fig. 2). Each motif ranged from 29 to 50 amino acids in length. Motif1-Motif10 are included in the members of Cl − /HCO 3 − exchangers and Na + / HCO 3 − cotransporters. Two genes in the Na + /Borate cotransporter group (slc4a11a, slc4a11b) contained only seven conserved motifs, excluding Motif3, Motif4 and Motif7. Motifs 1, 2, 5, 6, 8, 9, and 10 were shared by 15 slc4 genes, suggesting that they are located in the main functional domain of slc4 genes, namely, the HCO 3 _cotransp domain that is responsible for the transport of anions. Overall, genes from the same transporter group showed similar motif characteristics, and the distribution of motifs was consistent with the results of the phylogenetic analysis.

Phylogenetic analysis and copy number analysis of slc4 genes
In this study, 202 amino acid sequences of the slc4 genes of 15 representative vertebrates (4 of which belong to the genus Triplophysa) were selected to construct a phylogenetic tree. Table 2 summarizes the copy numbers of slc4 gene members in T. dalaica and other vertebrates. It can be seen that the selected species range from higher vertebrates to teleost fishes, and the number of slc4 genes varies from 10 to 15. All slc4 genes in selected in higher vertebrates (human, mouse, Xenopus laevis) are singlecopy, and duplicate slc4 gene copies are present in common teleost genomes. The above results indicate that the slc4 gene family expanded significantly during the evolution of teleost fishes. Furthermore, the slc4a9 gene is not found in all teleosts.
Phylogenetic analysis can provide a basis for the annotation of slc4 genes in T. dalaica. According to the gene clustering results shown in Fig. 3, all slc4 genes were divided into three categories: Cl − /HCO 3 − exchangers, Na + /HCO 3 − cotransporters, and Na + /Borate cotransporters. Genes within these three categories were further divided into 10 separate clades. The slc4 genes (red stars) of the T. dalaica were in the corresponding clade with other species and obtained a high support rate (boostrap value). The slc4 genes of a total of four fish species in the Triplophysa genus were clustered with orthologous genes of other species, especially zebrafish, indicating that they have been highly conserved in the evolutionary process. The clustering results were consistent with those of previous studies [20,21].

Slc4 gene collinearity analysis and chromosomal location
Phylogenetic relationships supported the annotation of three single-copy genes (slc4a3, slc4a7, and slc4a8). To confirm the evolutionary relationship of the slc4 genes with multiple copies (slc4a1, slc4a2, slc4a5, slc4a7, slc4a10, and slc4a11) and to provide more evidence for annotation, the adjacent genetic regions of the slc4 genes were analyzed for collinearity (Fig. 4). The results showed that the slc4 genes in T. dalaica had the highest collinearity with the zebrafish genes, and that they shared a conserved genomic neighborhood with zebrafish and Nile tilapia, confirming the reliability of the slc4 genes annotation. Therefore, our findings not only well support the annotation of the slc4 genes in T. dalaica, but again show that the slc4 genes have been highly conserved during evolution. In addition, this study found that the 15 slc4 genes of T. dalaica were distributed on 13 chromosomes (Fig. 5). Two of the slc4 genes (slc4a5b, slc4a4a) were located on chromosome 4; slc4a10b and slc4a3 were located on chromosome 8, and the other 11 genes were located on different chromosomes. Compared with higher vertebrates, gene duplication is more common in teleost fishes. Therefore, based on the chromosomal collinearity analysis and the location information of each slc4 gene, we speculate that the duplicated slc4 genes in T.   Fig. 1. All motifs were identified from the MEME database, and different colored blocks represent different motifs. b Motifs of T. dalaica slc4 proteins dalaica were primarily due to a teleost-specific whole genome duplication (WGD) event [22].

Recombinant analysis of slc4 family genes
Genetic recombination plays an important role in the evolution of gene families [23]. Only one gene had a recombination signal (Table 3). Slc4a2a has a recombination site with slc4a1b at 2554 ~ 2892 bp. Therefore, in order to adapt to environmental changes, T. dalaica increased the number of slc4 genes and the expression of slc4 genes through intragene recombination.

Analysis of the expression results of slc4 genes
Quantitative polymerase chain reaction (qPCR) was used to analyze the expression of slc4 genes in different tissues (Fig. 6, Supplementary Fig. S1). The differential expression of slc4 genes and the regulation mode of adapting to high salinity environments were visually displayed. In different habitats, the coping mechanisms of slc4 genes differ, and thus their expression levels may be different. The slc4 genes show tissue-specific expression patterns, and they play major regulatory roles in the brain, liver, gill, kidney, and other tissues. Differential expression patterns of slc4 genes have been observed in different tissues in freshwater and alkaline water habitats. For example, slc4a4a, slc4a7, and slc4a10a are highly expressed in brain tissue, but are expressed at lower levels in kidney. For osmoregulatory organs, slc4a1b, slc4a7, and slc4a4b were highly expressed in the gills. In the kidney, slc4a1a, slc4a3, and slc4a10b were highly expressed. Different copies of the same gene also showed different expression patterns in different tissues. For example, relative to the freshwater group, slc4a1a was only highly expressed in the spleen and kidney, while slc4a1b was expressed at a low level in these two tissues. This indicates that the two slc4a1 gene copies have different physiological functions in T. dalaica. From freshwater to alkaline water environments, some genes were upregulated, and others were downregulated. For example, slc4a4b was highly expressed in the liver in freshwater environments, but downregulated in alkaline water environments. Slc4a10b was upregulated in the brain, indicating that it has an important regulatory role in adaptation to highly alkaline environments.

Characteristics of slc4 gene family members of T. dalaica
The slc4 gene family members are membrane transporters, and previous studies have shown that this gene family is involved in the transport of HCO 3 − in mammals [24,25]. In recent years, studies have shown that the slc4 genes also play important roles in teleost fishes, and findings in zebrafish, Leuciscus waleckii, and Oreochromis niloticus suggest that some genes are involved in ion osmosis and intracellular physiological processes such as pH regulation [10][11][12]14]. At present, there are few systematic studies on the entire slc4 gene family. To have an overall understanding of the functions of the slc4 gene family, the members of the family in T. dalaica were systematically analyzed by combining bioinformatics technology and real-time PCR.
In this study, a total of 15 slc4 genes were identified in the T. dalaica genome distributed on 13 chromosomes, and there was no tandem duplication of the slc4 gene copies (Fig. 5). According to the existing research, the gene family members are divided into three different branches according to their functions in ion transport. By aligning the amino acid sequences of multiple species and constructing a phylogenetic tree (Fig. 3), we found that the 15 sequences were divided into 10 branches (slc4a1, slc4a2, slc4a3, slc4a4, slc4a5, slc4a7, slc4a8, slc4a9, slc4a10, and slc4a11). Orthologous slc4 genes from all species clustered together. The corresponding slc4 genes of T. dalaica were first clustered with three species of Triplophysa; second, the sequences clustered with those of zebrafish. Combined with the results of the collinearity comparison of the adjacent genes of T. dalaica, zebrafish, and Nile tilapia (Fig. 4), this demonstrates that the slc4 gene family is relatively conserved. It is worth noting that the slc4 genes differ in the evolution of higher vertebrates and teleost fishes, and the phenomenon of gene deletion has been accompanied by whole genome duplication [26]. This phenomenon may be due to gene loss during evolution and may reflect ecological adaptations in teleosts and other vertebrates [27,28].

Expression patterns of members of the slc4 family in T. dalaica
Although there are structural similarities among the slc4 gene family members, there are different expression patterns in different tissues. In this study, qPCR showed that the slc4 genes were expressed in the brain, spleen, gill, liver, gonad, and kidney of T. dalaica. T. dalaica from Anyang and Inner Mongolia showed differences in their ability to regulate carbonate-alkaline metabolism due to long-term adaptability in water environments with different alkalinities. The above differences were reflected in the following aspects: Five members identified in this study (slc4a1a, slc4a1b, slc4a2a, slc4a2b, and slc4a3) belong to Cl − /HCO 3 − exchangers that exchange monovalent anions from opposite sides of the cell membrane. The gene slc4a1, a Cl − / HCO 3 − exchanger for erythrocytes, was one of the earliest identified transporters among the members of the slc4 gene family. Guizouarn et al. found through experiments that slc4a1 exhibits different transport characteristics in response to different stimuli [29]. Slc4a1a mediates Cl − and HCO 3 − exchange in the kidney, and it is also required for normal acidification of urine. Studies have shown that the expression level of slc4a1b in zebrafish gills is lower in high Na + environments than in low Na + environments, but higher in acidic water than in freshwater environments [21]. Shmukler et al. found that the expression levels of slc4a2 genes were distinct in different developmental stages and tissues of the zebrafish [9]. Previous studies have found that slc4a3 exists in mammalian excitable tissues such as brain [30,31], heart [32,33], and epithelial cells of the kidney and gastrointestinal tract. There is currently evidence that the zebrafish slc4a3 gene is sensitive to pH changes; at a pH of 8.5, the anion exchange activity is significantly increased [34]. It has been hypothesized that slc4a3 regulates pH by exporting HCO 3 − upon intracellular alkali loading. In our study, the slc4a1a gene expression in the kidney and spleen of T. dalaica in a saline-alkali fish was significantly higher than that in freshwater individuals. In T. dalaica, the expression of slc4a1b in the gill was much lower in the saline-alkali environment than in the freshwater environment, and the expression of slc4a1b gene in the brain, liver, and gonad was significantly increased in the saline-alkali environment. The expression patterns of slc4a2 genes in different tissues of the two habitats were basically the same as that of slc4a1. Slc4a3 was expressed to varying degrees in the brain and gonads. However, the role of slc4a3 in mediating anion exchange in teleost development and physiology remains to be elucidated [34].
(2) Na + /HCO 3 − cotransporters Members of this branch transport Na + and HCO 3 − from one side of the cell membrane to the other [5]. According to whether the protein is electrogenic during transmembrane transport, these genes can be divided into two categories, one being electrogenic (slc4a4 and slc4a5), the other being electrically neutral, including slc4a7, slc4a8, and slc4a10 [35]. Slc4a4 genes are essential for the acid-base balance of various tissues of the body. Previous studies have shown that slc4a4 genes are highly expressed in the gills, kidneys, intestines, and livers of freshwater fishes such as Oncorhynchus mykiss [36][37][38]. Slc4a4 genes are responsible for pH regulation in the liver, whereas in the kidney, slc4a4 genes play key roles in the reabsorption of HCO 3 − . Moreover, the slc4a4 genes are also expressed in gills of Anguilla japonica [39] and the intestines of Takifugu obscurus [13] during seawater-acclimating processes. The latter study found that slc4a4 gene expression was detected in the retina, pronephros, and gills of zebrafish. The gill has a mechanism to prevent metabolic acidosis and alkalosis. Slc4a4 gene expression in zebrafish gills is consistent with its function observed in freshwater and marine fishes, where it is responsible for regulating osmotic pressure and pH in biological corpora [40]. It has been speculated that the systemic acid-base balance of fish is likely to be accomplished by the joint action of kidneys and gills. More studies have found that slc4a4 gene is widely expressed in all cells in the zebrafish ventricle, and it has been speculated that it may be involved in absorption from the cerebrospinal fluid to the brain parenchyma [41,42]. Similarly, slc4a8 genes were found to contribute to cerebrospinal fluid secretion in mice   [43], and human slc4a8 mRNA exists in multiple parts of the human brain, testis, kidney, and ovary. The mRNA encoding slc4a10 genes was originally found in rat brain, pituitary, testis, kidney, and ileum [44]. Our findings are largely consistent with those of previous studies. The expression of slc4a4 genes in the gill, liver, and brain of T. dalaica in saline-alkali waters was significantly increased, indicating that the fish maintain ion homeostasis and acid-base balance in the body in response to a strong saline-alkali environment. The expression levels of these genes were significantly enhanced to prevent alkalosis [45]. At present, there are almost no reports or studies on slc4a5 and slc4a7 in teleosts. We speculate that the slc4a7 genes may play a greater role in gill tissue, because the expression of these genes was significantly increased in saline-alkali waters. In addition, the slc4a7 gene was also expressed in the brain, liver, and gonad tissues. Therefore, further research is needed on the expression patterns of slc4a5 and slc4a7 involved in acid-base regulation in vivo and their corresponding functions [46]. When fish were exposed to high alkalinity, slc4a10b was expressed at higher levels in the brain than in freshwater environments. In addition, the slc4a10 genes were significantly differentially expressed in the brain, kidney, and gonad tissues of T. dalaica under saline-alkali stress, indicating that there may have been functional differentiation.
(3) Na + /Borate cotransporters The Na + /Borate cotransporter is a mixed cotransporter. The Na + /Borate cotransporter can transport Na + and two HCO 3 − , one Na + and one CO 3 2− , or one NaCO 3 − ion pair into the cell while exchanging one Cl − . In the presence of borate, slc4a11 genes are voltage-regulated Na + /Borate transporters, whereas in the absence of borate, slc4a11 genes mediate Na + and OH − (H + ) transport [7]. The current study found that slc4a11 genes were required for renal urine concentration, but the link between the Na + /Borate cotransporter in animals and humans is unclear [47]. Our results suggest that in the brain, gills, and gonads, slc4a11 in T. dalaica may be involved in the regulation of ion and acid-base/nitrogen homeostasis.
The T. dalaica in Dali Lake have a strong alkali tolerance due to long-term habitation of water with high carbonate alkalinity. The T. dalaica in Anyang city can also live normally in fresh water. This indicates that the fish have evolved multiple regulatory modes under different alkalinity environments, suggesting a potential breeding Fig. 6 Expression of slc4 genes in T. dalaica in various tissues in different habitats object in saline-alkali areas for aquaculture. The slc4 genes of T. dalaica are upregulated or downregulated by environmental salinity to maintain survival, growth, and reproduction, and thus form a characteristic of this species to adapt to different environmental conditions. At present, this gene family has been studied in mammals, and the expression patterns of these transporters in teleost fishes have a certain similarity with those of mammals.

Conclusions
In this study, 15 slc4 genes were identified in the T. dalaica genome, and their basic information of sequence features, conserved domains, and conserved motifs were statistically analyzed. The 15 slc4 genes were distributed on 13 chromosomes. A phylogenetic tree was established using the amino acid sequences of the slc4 genes of representative vertebrates, and the tree was divided into three categories according to the different transport ions. We discussed the evolutionary relationships of the slc4 genes in T. dalaica and found that duplicate copies of the slc4 genes were present in common teleost genomes due to the WGD event in teleosts. The results show that the slc4 gene family expanded significantly during the evolution of teleost fishes. The results of a collinearity analysis indicated that the slc4 genes in T. dalaica had strong collinearity with those of zebrafish, and the slc4 genes were highly conserved in the evolutionary process. This finding further supports the annotation results for the T. dalaica genome. The differential expression of slc4 gene family members in six tissues including gill, kidney, and brain under different habitats was analyzed by RT-PCR. We speculated that slc4a1, slc4a4, and slc4a7 genes may play greater roles in the gill. This study will help us to understand the expression patterns of slc4 gene family members in saline-alkali environments and will provide support for subsequent studies on how slc4 genes regulate fish adaptation to saline-alkali environments. This will aid the search for suitable breeding varieties in saline-alkali areas for aquaculture.

Identification of slc4 gene family members
The CDS sequences and amino acid sequences of the zebrafish slc4 genes were obtained from the Ensembl (http:// asia. ensem bl. org) database, and the protein sequences of 14 zebrafish slc4 genes were used as the query sequences. BLAST (1e-5) was performed in the genome database of T. dalaica, and the National Genomics Data Center under Accession number PRJNA624716. The extracted candidate genes of T. dalaica were subjected to reverse blast at NCBI to verify the accuracy.

Slc4 genes structure analysis
Based on the amino acid sequences of the slc4 genes of T. dalaica, the SMART 7.0 (http:// smart. emblheide lberg. de/) tool was used to predict the conserved domain structures of the slc4 genes, and the results were further confirmed by combining with the conserved domain prediction in NCBI. ExPASy (https:// www. expasy. org/) was used to predict the isoelectric point and molecular weight of each slc4 protein. .0 [48] (http:// www. csbio. sjtu. edu. cn/ bioinf/ Cell-PLoc-2/) was used to predict the subcellular localization of slc4 gene products. The location information of the slc4 genes and the structural information of exons and introns were obtained from the annotation file of the T. dalaica reference genome (GFF) and visualized using TBtools [49] software. The motifs of the identified slc4 proteins were predicted using the MEME (http:// meme-suite. org/) program. The maximum value of the searched motifs was set to 10; the minimum width was set to 6, and the maximum value was 50. The results were visualized in TBtools [49].

Phylogenetic analysis of slc4 genes
To further understand the phylogenetic relationships and nomenclature of the slc4 genes, the CDS sequences and amino acid sequences of some representative species' slc4 genes were downloaded from the NCBI and Ensembl databases. These species were the zebrafish (Danio rerio), human (Homo sapiens), mouse (Mus musculus), clawed frog (Xenopus laevis), stickleback (Gasterosteus aculeatus), European sea bass (Dicentrarchus labrax), channel catfish (Ictalurus punctatus), large yellow croaker (Larimichthys crocea), Nile tilapia (Oreochromis niloticus), Japanese medaka (Oryzias latipes), and fugu (Takifugu rubripes). The genomes of these species have been well annotated. The published genome data of three species of Triplophysa were downloaded from the Ensembl database and Gigabase; these were Triplophysa siluroides, Triplophysa tibetana, and Triplophysa bleekeri. The identification of the slc4 family members of these three species was similar to that for Triplophysa dalaica. Using the slc4 protein sequences of zebrafish as the query sequences, the three candidate slc4 genes of Triplophysa were searched by BLASTP [49] and then combined with NCBI to perform a reverse blast to obtain the final confirmed slc4 genes.
The amino acid sequences of 202 slc4 genes of 15 species obtained above were aligned by ClustalW2 [50] (http:// www. ebi. ac. uk/ Tools/ msa/ clust alw2/) using the default parameters. Based on the neighbor-joining method, the aligned sequences were employed in MEGA 7.0 [51] to construct a phylogenetic tree, and the Jones-Taylor-Thornton (JTT) model was selected with a bootstrap value of 1000. The evolutionary tree was further polished via the iTOL (https:// itol. embl. de/ itol. cgi) online site.

Collinearity analysis and recombination events detection in slc4 genes
Collinear analysis was conducted to compare the neighbored genetic regions of slc4 genes among zebrafish, Nile tilapia, and T. dalaica. The chromosomal locations of zebrafish and Nile tilapia slc4 family members and their adjacent genes were identified using Genomicus 97.01 (https:// www. genom icus. biolo gie. ens. fr/ genom icus-97. 01/ cgi-bin/ search. pl). The locations of members of the slc4 family in T. dalaica were derived from the results of the genome assembly annotations. The three species were mapped for collinearity based on the obtained genetic information. To further understand the evolutionary feature of slc4 genes in the T. dalaica. Potential recombination events were identified using RDP v4.8 [52]. GENECONV [53] were used to analyze the sequences with 0.05 P value cutoff and 100 permutation parameters in the study.

Acquisition of experimental fish
The experimental T. dalaica used in this study came from two habitats, Lushui River (fresh water) in Linzhou, Anyang (35°54′28.0″N, 113°51′26.0″E), and Dali Lake (saline-alkali water) in Inner Mongolia. Three samples were taken from each group. Each animal was euthanized with buffered tricaine methanesulfonate (MS-222). The six tissues of brain, liver, spleen, gonad, kidney, and gill were taken, and the tissues were placed in 400 μL RNAiso Plus (Trizol, Takara) in 1.5 ml enzyme-free centrifuge tubes. After being kept at 4 °C for 4 h, samples were stored in a − 80 °C refrigerator.

Total RNA extraction, cDNA synthesis, and quantitative real-time PCR (qPCR) analysis
The RNA extraction method referred to the instructions of tissue preservation reagent RNAiso Plus. Extracted RNA was tested for concentration and integrity. Reverse transcription of qualified RNA was performed for each sample following the steps in the HiScript II Reverse Transcriptase manual. Primer 5.0 was used to design specific primers for the members of the T. dalaica slc4 gene family (Table S1). The expression of slc4 genes of T. dalaica in different habitats and different tissues was detected by qRT-PCR. The qPCR was conducted according to the instructions for ChamQ Universal SYBR qPCR Master Mix reagent. The total reaction volume was 10 μl, comprising 5 μl of 2 × ChamQ Universal SYBR qPCR Master Mix, 0.2 μl of forward primer, 0.2 μl of the reverse primer, 1 μl template cDNA, and 3.6 μl ddH 2 O. The reaction conditions were as follows: pre-denaturation at 95 °C for 30 s followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and a final extension at 72 °C for 2 min. The freshwater habitat group was used as the control group; the saline-alkali water group was used as the treatment group, and β-actin was used as the internal reference gene. The method of 2 -ΔΔCt [54] was used for data processing, and the relative expression levels were calculated. Data were normalized in TBtools [48] and the results were visualized as heatmaps to compare the expression of each gene in different tissues. The relative expression of slc4 gene family in the two habitats was statistically graphed in the study.